Glycan and Glycopeptide Analysis
In the area of biotherapeutics, recombinant monoclonal antibodies (mAbs) have gained significant
importance in diagnostic and therapeutic applications in recent years. The general structural features of
mAbs, such as assembly of light and heavy chains via disulfide bridges, are commonly known. However,
the heterogeneity of antibodies, mostly due to the variation of attached sugar moieties, requires thorough
characterization. These molecules contain complex oligosaccharide moieties whose sites of attachment,
presence or absence, and relative profiles can have a significant impact on therapeutic efficacy,
pharmacokinetics, immunogenicity, folding, and stability of a biologic drug. Thorough characterization is
necessary to verify the correctness of the overall molecule and provide a reproducible, safe, and effective
biological drug compound.
Mass spectrometry (MS) has emerged as one of the most powerful tools for the structural elucidation of
glycans. This is due to its sensitivity of detection and its ability to analyze complex mixtures. The complex
branching and isomeric nature of glycans pose significant analytical challenges to the identification of
these structures. In addition to the characterization of the sugar sequence, the analysis must elucidate
branching, linkages between monosaccharide units, and the location of possible sulfate or phosphate
groups. To ensure comprehensive structural elucidation and structural isomer differentiation, a
combination of high mass resolution, accurate mass, sensitivity, multistage fragmentation, and multiple
fragmentation techniques are required.
The high-resolution accurate mass (HRAM) of the Orbitrap family of mass spectrometers enables the
rapid discovery of glycan and glycopeptide information that was previously difficult or impossible to
capture. The Thermo Scientific
™
Q Exactive
™
MS enabled with multiple fragmentation techniques such
as HCD and ETD is the most versatile MS/MS system in the modern laboratory today.
The combination of high resolution chromatography and new separation technologies specific for
glycans, such as the Thermo Scientific
™
GlycanPac
™
AXH-1columns further drives a comprehensive
LC-MS integrated workflow for glycan and glycopetide analysis.
